Human factor XIII as a normalization control for immunoassays

ABSTRACT

The present disclosure provides compositions and methods that are useful for normalizing the amount of signal detected in an assay, such as an immunoassay. The compositions and methods are useful for improving the accuracy of immunoassays, such as immunoassays that detect whether a subject is infected with a retrovirus such as HIV.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application claims benefit of priority to U.S. ProvisionalPatent Application No. 61/663,293, filed Jun. 22, 2012, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention resides in the field of immunodiagnostics.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The Sequence Listing written in file095191-094510US-0877710_SequenceListing.txt created on Nov. 9, 2016,2,025 bytes, machine format IBM-PC, MS-Windows operating system, inaccordance with 37 C.F.R. §§1.821- to 1.825, is hereby incorporated byreference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Immunoassays that detect HIV antigens and antibodies directed againstHIV antigens in biological samples are useful to determine if a subjectis infected with or has been exposed to HIV. Current assays detect HIVantigens and antibodies to HIV-1, including groups O and M, andantibodies to HIV-2. However, assays that detect the amount of analytesin a biological sample are subject to errors that can produce spuriousresults. There are numerous factors that can produce errors. Thesefactors include variations in reagent concentration (e.g., due toreagent stability or instrument pipetting errors), instrument processingsteps (e.g., reagent dispense volumes, incomplete aspiration after washsteps resulting in reagent dilution, incubation timing), instrumentconditions (detector temperature), and sample matrix effects (e.g. serumvs. plasma).

One method for verifying the amount or volume of a sample that isanalyzed in an assay is described in U.S. Pat. No. 7,141,362. The methoddetects the amount of human blood coagulation Factor XIII (hFXIII)subunits a and b in a sample to determine the volume of a sample, andcan also be used to distinguish plasma and serum from other types ofbiological samples. The amount of hFXIII in a sample is detected in animmunoassay using antibodies to hFXIII subunits immobilized on solidsupports.

Further, replicate analysis of samples such as calibrators, controls orpatient specimens (serum or plasma) can produce inconsistent resultsleading to high coefficients of variation (percent relative standarddeviation). In some cases, the variation can be as high as 50% or moreand can be sufficient to produce a response classified as positive forsome replicates but negative for others. This variability isunacceptable for critical assays such as HIV detection. This inventionaddresses errors in analysis that lead to high coefficients ofvariation.

SUMMARY OF THE DISCLOSURE

The present disclosure describes a normalization factor that can be usedas a normalization standard and/or internal control in a biologicalassay. In some embodiments, the normalization standard is attached to asolid support that is included in the assay. In one aspect, a method fornormalizing results in an immunoassay for the detection of analytes in abiological sample is described. The analytes are detected by binding tobinding members that specifically bind an individual analyte. Byincluding a normalization factor in the assay, errors introduced byvariations in sample processing or biological matrix effects associatedwith various sample types can be corrected. In some embodiments, thenormalization factor does not specifically bind to an analyte in thesample.

Thus, in some embodiments, the biological sample is incubated with oneor more binding members which bind one or more of the analytes in thesample, and detecting whether any of the analytes bind to the bindingmembers. For example, if the analyte is an antigen, the correspondingbinding member can be an antibody that specifically binds the antigen.Likewise, if the analyte is an antibody, the corresponding bindingmember can be an antigen that specifically binds the antibody. Thus, anindividual target analyte specifically binds to a corresponding bindingmember, and a panel of analytes can bind to a corresponding panel ofbinding members. The analytes that bind to a given binding member arethen identified. One means of such identification is to use bindingmembers that are immobilized on a solid support, with a different solidsupport for each binding member. For example, molecules of only onebinding member are immobilized on an individual solid support, and thesolid supports for different binding members are distinguishable fromeach other by means other than the binding members themselves. The solidsupports can be distinguished from each other by using differentiationparameters associated with the solid supports, all supports bearing anyone binding member being differentiable from all supports bearing any ofthe other binding members by the differentiation parameters. Thus, thedifferentiation parameters can be used to divide the solid supports intosubpopulations that are differentiable from each other.

The detection of immunological binding between analytes from the sampleand the binding members can be achieved by the use of binding agents.For example, binding agents having binding affinity for thenormalization factor and binding agents having binding affinity for eachof the analytes in the sample can be used to detect binding. In certainembodiments, the binding agents are incubated with the solid supportshaving binding members immobilized thereon under conditions that promotebinding of the binding agents to the normalization factor and theanalytes. In some embodiments, the binding agent is conjugated to alabel. In some embodiments, the label is biotin. In some embodiments,the label is a detectable label, such as a fluorescent moiety.

Following incubation of the binding agents with the solid supports, thesolid supports are recovered to separate the supports having theanalytes and/or normalization factor bound thereto from unbound bindingagents. In embodiments where the binding agent is conjugated to biotin,the solid supports can be incubated with streptavidin that is coupled toa detectable label. In some embodiments, the detectable label is afluorescent label, such as phycoerythrin (PE). The amount of label boundto the solid supports is then detected. For example, the label can bedetected by any method capable of measuring the amount of fluorescentsignal emitted by the label.

The amount of label detected can be correlated with the differentiationparameters to obtain values that are representative of the level oramount of each individual analyte in the sample, as well as values thatare representative of the level or amount of the normalization factorthat is bound to the solid support. This allows the valuesrepresentative of the levels of each individual analyte in the sample tobe normalized to the value representative of the level of thenormalization factor. The normalization allows variations in sampleprocessing to be corrected, thereby improving the performance of theassay. The normalization also allows for variations in signal intensity,for example, from the detectable label, to be corrected, where thevariation in signal intensity is due to the source of the sample (i.e.,biological matrix effects).

In one embodiment, the normalization factor is human Factor XIII(hFXIII). In one embodiment, the method uses human Factor XIII coupledto a solid support as a normalization factor and/or internal control ina biological assay. Thus, in certain embodiments, one subpopulation ofsolid supports has hFXIII immobilized thereon. In some embodiments, thesolid support is a bead or magnetic bead. In one embodiment, hFXIII isimmobilized to a bead. In some embodiments, hFXIII is immobilized to amagnetic bead. In one embodiment, hFXIII immobilized to a bead ormagnetic bead is referred to as a Signal Normalization Bead (SNB). ThehFXIII bound to the solid support can be detected using an anti-hFXIIIantibody. The anti-hFXIII antibody is typically not present in thebiological sample isolated from the subject, but is added with otherbinding agents specific for analytes during sample processing in orderto provide a means for detecting the hFXIII immobilized on the solidsupport, as described herein.

In some embodiments, one subpopulation of solid supports has antibodiesto hFXIII immobilized thereon, which are used to detect binding ofhFXIII (i.e., subunits a and/or b) present in the biological sample, asdescribed in U.S. Pat. No. 7,141,362, which is incorporated by referenceherein in its entirety. Thus, in some embodiments, anti-hFXIIIantibodies are immobilized on a solid support comprising a bead ormagnetic bead. In one embodiment, the bead or magnetic bead havinganti-hFXIII antibodies immobilized thereon is referred to as a SerumVerification Bead (SVB). The SVB allows for confirmation that a serumsample was present in the assay.

In another aspect, a method for normalizing assay results is provided,such as a multiplex immunoassay for the detection of analytes in abiological sample. In certain embodiments, the immunoassay is for thedetection of a panel of analytes whose levels are indicative ofinfection with human immunodeficiency virus (HIV) in a biologicalsample. The panel of analytes are detected by binding to a correspondingset or panel of binding members that each individually bind anindividual analyte. Binding of one or more analytes in the sample to oneor more binding members indicates that the biological sample is from asubject infected with HIV. By including a normalization factor, errorsintroduced by variations in sample processing or variations due tobiological matrix effects can be corrected.

In some embodiments, the members of the panel of analytes are HIVantigens and/or antibodies that bind to HIV-1 and HIV-2. In certainembodiments, the members of the panel of analytes include:

(i) p24 antigen,

(ii) antibodies to HIV Type 1,

(iii) antibodies to HIV Type 2,

(iv) antibodies to HIV Type 1, group O, and

(v) antibodies to HIV Type 1, group M.

In some embodiments, the binding member specifically binds to one memberof the panel of HIV analytes described above. In some embodiments, thebinding member is an anti-p24 antibody, HIV-1 envelope protein gp-160,SPOH, or AFR.

The immunoassay can also include a solid support having an antibody tohuman Factor XIII immobilized thereon. As described above, the antibodyto human Factor XIII binds to hFXIII present in the biological sample,and thus serves as an assay to verify that the sample contained serum orplasma, helping to avoid false negative results. Thus, in oneembodiment, the binding member is an antibody to human Factor XIII.

The binding of the panel of analytes whose levels are indicative ofinfection with HIV can be detected using biotinylated conjugates. Insome embodiments, the biotinylated conjugates bind to HIV antigens,antibodies to HIV-1, and antibodies to HIV-2. In some embodiments, thebiotinylated conjugates bind to a normalization factor. In someembodiments, the normalization factor is hFXIII.

In some embodiments, the biotinylated conjugates are selected from39-PFN-BgG, SV2V, AFR, antibodies to p24 antigen, and antibodies tohuman Factor XIII.

In some embodiments, the solid support is a bead or a magnetic bead.

In some embodiments, the solid support is coupled totetramethylcadaverine rhodamine (TMRC).

The invention further resides in a kit that includes a normalizationfactor immobilized on a solid support, and a panel of binding members,each of which is immobilized on a solid support. The panel of bindingmembers includes any of the various binding members presented above, andthe solid supports further contain differentiation parameters that areselected such that all of the supports bearing any one binding member ofthe panel are differentiable by these parameters from all of thesupports bearing other binding members of the panel.

In some embodiments, the kit includes a panel of binding members fordetermining whether a subject is infected with HIV or for determiningthe stage of infection in the subject. In some embodiments, the subjectis a human.

In certain embodiments, the kit further provides tetramethylcadaverinerhodamine (TMRC) immobilized on a solid support.

DEFINITIONS

A “binding agent” is a compound having binding affinity for anothercompound. For example the binding agent can bind with high affinity tothe normalization factor that is immobilized on a solid support. Thebinding agent can also bind to an analyte in the biological sample. Thebinding agent can be immunoreactive with the normalization factor or atarget analyte. For example, the binding agent can be an antibody thatspecifically binds the normalization factor or an antigen in thebiological sample, or the binding agent can be an antigen thatspecifically binds an antibody in the biological sample. The bindingagent can be conjugated to a label that allows the binding agent to bedetected. In some embodiments, the binding agent is not immobilized on asolid support.

The term “binding member” is used herein to denote a molecule or agentthat specifically binds to a target analyte. The binding member can beimmunoreactive with a target analyte. For example, the binding membercan be an antigen that specifically binds to an antibody in thebiological sample, or the binding member can be an antibody thatspecifically binds to an antigen in the biological sample. The bindingmember can be immobilized on a solid support, including but not limitedto a bead or magnetic bead.

A “normalization factor” is a molecule that can be used to normalize anassay result. The normalization factor typically does not react with orbind with high affinity to an analyte of interest in the sample.

The term “label” or “detectable moiety” is used herein to denote acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. Examples of labelsare 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., ascommonly used in an ELISA), biotin, digoxigenin, and haptens andproteins or other entities which can be made detectable, e.g., byincorporating a radiolabel into the peptide or by being used to detectantibodies specifically reactive with the peptide. The labels can beincorporated, for example, into antibodies and/or other proteins at anyposition. Any method known in the art for conjugating the antibody orprotein (peptide) to the label can be employed, for example, usingmethods described in Hermanson, Bioconjugate Techniques 1996, AcademicPress, Inc., San Diego. Alternatively, methods using high affinityinteractions can achieve the same results where one of a pair of bindingpartners binds to the other, e.g., biotin and streptavidin. The proteinsof the invention as described herein can be directly labeled as withisotopes, chromophores, lumiphores, chromogens, or indirectly labeledsuch as with biotin to which streptavidin in a complex with afluorescent, radioactive, or other moiety that can be directly detectedcan then bind. Thus, a biotinylated antibody is considered a “labeledantibody” as used herein.

The term “antibody” as used herein refers to a polypeptide encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin light chains are classified as either kappa or lambda.Immunoglobulin heavy chains are classified as gamma, mu, alpha, delta,or epsilon, which in turn define the immunoglobulin classes, IgG, IgM,IgA, IgD and IgE, respectively.

An example of a structural unit of immunoglobulin G (IgG antibody) is atetramer. Each such tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(VL) and “variable heavy chain” (VH) refer to these light and heavychains, respectively.

Antibodies exist as intact immunoglobulins or as well-characterizedfragments produced by digestion of intact immunoglobulins with variouspeptidases. Thus, for example, pepsin digests an antibody near thedisulfide linkages in the hinge region to produce F(ab′)2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab′)2 dimer can be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab′)2dimer into two Fab′ monomers. The Fab′ monomer is essentially an Fabwith part of the hinge region (see, Paul (Ed.) Fundamental Immunology,Third Edition, Raven Press, NY (1993)). While various antibody fragmentsare defined in terms of the digestion of an intact antibody, one ofskill will appreciate that such fragments may be synthesized de novoeither chemically or by utilizing recombinant DNA methodology. Thus, theterm “antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or by de novo synthesisusing recombinant DNA methodologies such as single chain Fv.

The expression “specifically (or selectively)” in reference to bindingto an antibody, or “specifically (or selectively) immunoreactive with”or “having binding specificity for,” when referring to a protein,peptide, or antigen, refers to a binding reaction which is determinativeof the presence of the protein, peptide, or antigen in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein and do not bind in a significant amount to otherproteins present in the sample. Specific binding to an antibody undersuch conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raisedagainst a protein can be selected to obtain antibodies specificallyimmunoreactive with that protein and not with other proteins. A varietyof immunoassay formats may be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays, Western blots, or immunohistochemistry are routinely usedto select monoclonal antibodies specifically immunoreactive with aprotein. See, Harlow and Lane Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, NY (1988) for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity. Typically, a specific or selective reaction will be atleast twice the background signal or noise, and more typically more than10 to 100 times background.

Antibodies for use in certain embodiments of the present invention areanti-human antibodies, particularly those anti-human antibodies that arelabeled. Preferred among these anti-human antibodies are those that areantibodies to human IgG, those that are antibodies to human IgM, andthose that are antibodies to human IgA.

The term “biological sample” encompasses a variety of sample typesobtained from an organism. The term encompasses bodily fluids such asblood, saliva, serum, plasma, urine and other liquid samples ofbiological origin, solid tissue samples, such as a biopsy specimen ortissue cultures or cells derived therefrom and the progeny thereof. Asdescribed herein, typically, the biological sample will be a bodilyfluid or tissue that contains detectable amounts of an analyte ofinterest, e.g., an antigen, antibody, protein, peptide, nucleic acid, orsmall molecule. The term encompasses samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, sedimentation, or enrichment for certain components. Theterm encompasses a clinical sample, and also includes cells in cellculture, cell supernatants, cell lysates, serum, plasma, otherbiological fluids, and tissue samples. Preferred biological samples areblood samples, plasma samples, and serum samples.

The term “solid support” is used herein to denote a solid inert surfaceor body to which an agent, such as an antibody or an antigen, that isreactive in any of the binding reactions described herein can beimmobilized. The term “immobilized” as used herein denotes a molecularlybased coupling that is not dislodged or de-coupled under any of theconditions imposed during any of the steps of the assays describedherein. Such immobilization can be achieved through a covalent bond, anionic bond, an affinity-type bond, or any other chemical bond.

The term “particles” is used herein to denote solid bodies, often withlinear dimensions on the micron scale (i.e., less than 100 microns), ofany shape or surface texture. The term “beads” is used herein to denoteparticles that are spherical or near-spherical in shape, often polymericin composition.

“Multiplex” assays are analyses that simultaneously measure the levelsof more than one analyte in a single sample.

The term “analyte” or “target analyte” is used herein to denote amolecule that is present in a biological sample and is indicative of thepresence or absence of a biological condition in a subject, such as ahuman subject. The term biological condition is used without limitation,and can include both normal and abnormal conditions. For example, thebiological condition can be a pathological condition such as a disease,infection, cancer, or autoimmune disorder. The analyte can also indicatethe state or progression of a disease or infection, such as duringtreatment of the disease or during different stages of an infection. Theanalyte can also indicate a diagnosis or prognosis of a biologicalcondition.

The term “labeled binding moiety” refers to a molecule or reagent thatis conjugated or coupled to a label. The label can be a detectablelabel, such as a fluorophore, or can be indirectly labeled such as withbiotin or streptavidin, or with an enzyme that is useful for producing adetectable signal, such as alkaline phosphatase (AP) or horse radishperoxidase (HRP). The labeled binding moiety can bind to a binding agentor, where the label is avidin, streptavidin or an equivalent, to abiotinylated conjugate.

The term “differentiation parameter” is used herein to denote acharacteristic of the solid support that is independent of the bindingmember that is attached or immobilized to the solid support. Thus, adifferentiation parameter allows solid supports with different bindingmembers immobilized thereon to be distinguished from each other by meansother than the binding members themselves.

The term “biological matrix” refers to the source of the sample thatcontains the analytes of interest. For example, the biological matrixcan be a biological fluid such as, but not limited to, serum, plasma,whole blood, urine, saliva, lymph, or cerebral spinal fluid. The term“biological matrix effect” refers to the variation in the assay signalwhen the sample is derived from different biological matrices.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the use of a human Factor XIII coated bead as acontrol and to produce a normalized signal in a representative HIV assaythat detects HIV p24 antigen. Twelve (12) replicates were performed, andthe data was normalized as described in Example 1.

FIG. 2 illustrates the use of a human Factor XIII coated bead as acontrol and to produce a normalized signal in a representative multiplexHIV control assay that detects human polyclonal antibodies to HIV-1.Five (5) replicates were performed, and the data was normalized asdescribed in Example 2.

FIG. 3 illustrates the use of a human Factor XIII coated bead as acontrol and to produce a normalized signal in a representative multiplexHIV control assay that detects human polyclonal antibodies to HIV-2.Five (5) replicates were performed, and the data was normalized asdescribed in Example 2.

FIG. 4 illustrates the use of a human Factor XIII coated bead as acontrol and to produce a normalized signal in a representative multiplexHIV control assay that detects rabbit monoclonal antibodies to HIV-O.Five (5) replicates were performed, and the data was normalized asdescribed in Example 2.

FIG. 5 illustrates that the assay signal is decreased by various amountsof biotin conjugate remaining in the reaction vessel in a representativeexperiment that detects antibodies to HIV-O, as described in Example 5.

FIG. 6 illustrates a plot of the relative response of the HIV p24antigen signal and human Factor XIII signal detected in serum and plasmasamples from the same donor in various biological matrices supplementedwith identical concentrations of HIV p24 antigen.

FIG. 7 illustrates a plot of the normalized signal from the sameexperiment as in FIG. 6, showing lower variation for all samples indifferent biological matrices.

DESCRIPTION OF SELECTED EMBODIMENTS

The present disclosure provides compositions and methods for correctingfor variations in sample processing and/or biological matrix effectswhen performing biological assays. The composition comprises anormalization factor coupled to a solid support. In some embodiments,the normalization factor can be any suitable molecule that is capable ofbeing attached to a solid support and that does not specifically bind toa target analyte in the biological sample. The normalization factor canbe covalently or non-covalently attached to the solid support. In someembodiments, the solid support is a bead or magnetic bead. Thus, in someembodiments, the normalization factor is coupled to a bead, and thenormalization bead interacts with reagents used during subsequentdetection steps of the assay in the same manner as the beads thatspecifically bind analytes from the sample. The solid support coupled toa normalization factor can be used to verify correct sample processingand for assay signal normalization. Signal normalization provides forimproved assay precision for biological samples, controls, andcalibrators, and improves signal stability over the lifetime of reagentsused in the assay.

In one embodiment, the normalization factor is human Factor XIII(hFXIII). In one embodiment, the method uses human Factor XIII coupledto a solid support as a normalization factor and/or internal control ina biological assay. In one aspect, a solid support coupled to hFXIII isprovided for use as a normalization standard and/or internal control.

Thus, in some embodiments, the hFXIII-coupled solid support can be usedin immunoassays to correct for variations in sample processing. In oneembodiment, the hFXIII-coupled solid support allows normalizing theamount of signal detected to improve the precision of the assay. Thesignal can be produced, for example, by a label. In one embodiment, thehFXIII-coupled solid support allows normalizing the amount of labeldetected in order to correct for loss of the amount of detectable labelduring storage of the reagents used in the method.

In some embodiments, the hFXIII-coupled solid support allows detectingan invalid assay, for example, by detecting when the assay signal fallsbelow a preset limit. For example, the signal for each sample can benormalized using hFXIII, and the normalized signal compared to expectedcontrol values. If the normalized sample signal is too low, then theassay results may be compromised due to matrix effects or otherprocessing error, and the results can be flagged as invalid.

The normalization factor coupled to a solid support can be used in amethod for analyzing a biological sample for the presence of one or moreanalytes whose levels are indicative of a disease state in a subject. Insome embodiments, the one or more analytes comprise a panel of analytes.Analytes of the present disclosure include antigens and antibodies thatare present in a biological sample from a subject, including but notlimited to a human subject. In some embodiments, the presence, absenceor the level of an analyte in a sample indicates that the subjectsuffers from or is at risk of developing a disease or other pathologicalcondition. For example, in some embodiments, the analytes indicate thatthe subject is infected with a virus, such as a retrovirus. Thus, insome embodiments, the analytes indicate that the subject is infectedwith human immunodeficiency virus (HIV), hepatitis C virus (HCV), orherpes simplex virus (HSV). In one embodiment, the analytes indicatethat the subject is infected with HIV. Analytes that are indicative ofHIV infection of a human subject include HIV antigens such as p24antigen, antibodies to HIV-1 (groups M, O, N and P), and antibodies toHIV-2 (including numerous subtypes).

In some embodiments of the method, a biological sample is incubated witha plurality of solid supports having binding members immobilized thereonfor each analyte in the panel. Binding members of the present disclosureinclude antigens and antibodies that are capable of specifically bindingto an analyte in the biological sample. For example, binding membersthat are capable of specifically binding to analytes indicative of HIVinfection of a human subject include anti-p24 antibodies, gp160 proteinfor detection of HIV-1, 39 PFN for detection of HIV-1/M, AFR fordetection of HIV-1/O, SPOH for detection of HIV-2, gp36 peptide fordetection of HIV-2, and SV2V for detection of HIV-2. 39 PFN and AFR arepeptides that mimic the immunodominant regions of HIV-1 groups M and O,respectively. SPOH and SV2V are peptides that mimic the immunodominantregions of HIV-2. The sequences of the peptides are provided in thefollowing table (Table 1).

TABLE 1 Sequences of peptides that bind HIV antibodies. HIV PeptidesBasic Cysteine Reactivity HIV-1/M HIV-1/O HIV-1/O HIV-2 HIV-2 Peptide39PFN Biotinyl-AFR21 AFR21K Biotinyl-SV2VPFVN 41SPOH PN 4052076 4055465701073 4055461 960453 #Amino 33 22 24 28 26 Acids MW 3704.4 2763.32793.3 3507.0 3054.5 SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4SEQ ID NO: 5 Sequence R Arginine R Arginine I Isoleucine V Valine LLeucine T Threonine A Alanine A Alanine V Valine I Isoleucine E GlutamicBiotinyl E Glutamic Acid Acid R Arginine K Lysine K Lysine K Lysine YTyrosine Biotinyl K Lysine Y Tyrosine Y Tyrosine L Leucine L Leucine LLeucine L Leucine L Leucine K Lysine N Asparagine N Asparagine QGlutamine Q Glutamine D Aspartic Q Glutamine Q Glutamine D Aspartic DAspartic Acid Acid Acid Q Glutamine Q Glutamine Q Glutamine Q GlutamineQ Glutamine Q Glutamine R Arginine R Arginine A Alanine A Alanine LLeucine L Leucine L Leucine R Arginine R Arginine L Leucine L Leucine LLeucine L Leucine L Leucine G Glycine N Asparagine N Asparagine NAsparagine N Asparagine I Isoleucine S Serine S Serine S Serine S SerineW Tryptophan W Tryptophan W Tryptophan W Tryptophan W Tryptophan GGlycine G Glycine G Glycine G Glycine G Glycine C Cysteine C Cysteine CCysteine C Cysteine C Cysteine S Serine K Lysine K Lysine A Alanine AAlanine G Glycine G Glycine G Glycine F Phenalanine F Phenalanine KLysine R Arginine R Arginine R Arginine R Arginine L Leucine L Leucine LLeucine Q Glutamine Q Glutamine I Isoleucine V Valine V Valine V ValineV Valine C Cysteine C Cysteine C Cysteine C Cysteine C Cysteine TThreonine Y Tyrosine Y Tyrosine H Histidine T Threonine T Threonine TThreonine T Threonine A Alanine S Serine S Serine T Threonine V Valine VValine V Valine V Valine P Proline P Proline F Phenalanine F PhenalanineN Asparagine V Valine N Asparagine

In some embodiments, the binding member is conjugated to anotherprotein, such as BSA, which is attached to a solid support. In oneembodiment, the binding member is an anti-hFXIII antibody. Thus, in oneembodiment, the biological sample is incubated with a plurality of solidsupports having an anti-hFXIII antibody immobilized thereon. Theincubation is performed under conditions that promote binding of eachanalyte, if present in the sample, to the binding members immobilized onthe solid supports. Conditions that promote binding of the analytes tothe binding members on the solid support include incubating the samplewith the solid supports in a buffered solution such as phosphate bufferat a pH of about 7.0 to about 7.4, at a temperature of about 25-37degrees C. for about 10 to 60 minutes. In some embodiments, the bindingconstitutes immunological binding.

The determination that immunological binding has occurred constitutesone or more steps in certain embodiments of this invention, and this caninvolve the separation or recovery of antigen-antibody complexes fromunbound antigen or antibody. One means of achieving such separation orrecovery is by the use of solid supports. Thus, the present disclosurefurther provides a plurality of solid supports having binding membersimmobilize thereon for each analyte of the panel of analytes. In someembodiments, the plurality of solid supports are divided intosubpopulations characterized in that each subpopulation comprises abinding member that binds to only one analyte of the panel of analytes.In some embodiments, each subpopulation of solid supports isdifferentiable from the other subpopulations of solid supports by adifferentiation parameter, as described herein.

In certain embodiments, there is provided a subpopulation of solidsupports having human Factor XIII immobilized thereon. In someembodiments, hFXIII is immobilized to a bead or magnetic bead. In oneembodiment, hFXIII immobilized to a bead or magnetic bead is referred toas a Signal Normalization Bead (SNB).

In certain embodiments, a subpopulation of solid supports has anantibody that binds hFXIII immobilized thereon. In some embodiments, theanti-hFXIII antibody is immobilized to a bead or magnetic bead. In oneembodiment, anti-hFXIII antibody immobilized to a bead or magnetic beadis referred to as a Serum Verification Bead (SVB). As described herein,the SVB can be used to verify the amount of biological sample added tothe assay, and whether the sample was from serum, plasma, or some otherbiological sample.

I. Solid Supports

Any type of solid support can be used in the invention. The solidsupport can comprise any surface capable of binding a protein, beingexposed to a sample, and separated into discrete populations. The solidsupport can be the wall or floor of an assay vessel, or a dipstick orother implement to be inserted into an assay vessel, or particles placedinside or suspended in an assay vessel. Particles, and especially beads,are particularly useful in many embodiments, including beads that aremicroscopic in size (i.e., microparticles) and formed of a polymericmaterial. Polymers useful as microparticles are those that arechemically inert relative to the components of the biological sample andto the assay reagents other than the binding members that areimmobilized on the microparticle surface. Preferred microparticlematerials, particularly when fluorescent labels are used in the assay,are those with minimal autofluorescence, and that are solid andinsoluble in the sample and in any buffers, solvents, carriers,diluents, or suspending agents used in the assay, in addition toallowing immobilization of the assay reagent. Examples of suitablepolymers are polystyrenes, polyesters, polyethers, polyolefins,polyalkylene oxides, polyamides, polyurethanes, polysaccharides,celluloses, and polyisoprenes. Crosslinking is useful in many polymersfor imparting structural integrity and rigidity to the microparticle.The size range of the microparticles can vary. In some embodiments, themicroparticles range in diameter from about 0.3 micrometers to about 100micrometers, and other embodiments, from about 0.5 micrometers to about40 micrometers, and in still other embodiments, from about 2 micrometersto about 10 micrometers.

In embodiments where the solid support is a bead, the biological samplecan be incubated with from about 0.1 μg to about 2.0 μg (microgram) ofbeads per sample in a reaction vessel. For example, the sample can beincubated with about 0.1 to about 2.0 μg, about 0.3 to about 1.7 μg,about 0.5 to about 1.5 μg, about 0.7 to about 1.2 μg, or about 0.8 toabout 1.0 μg of beads. In some embodiments, the amount of beadsincubated with the sample is sufficient to produce a concentration ofabout 0.002 micrograms/microliter (μg/μL) to about 0.04 μg/μL in areaction volume of about 50 μL. For example, the concentration can rangefrom about 0.002 to about 0.040 μg/μL, about 0.004 to about 0.035 μg/μL,about 0.008 to about 0.030 μg/μL, about 0.010 to about 0.025 μg/μL,about 0.010 to about 0.020 μg/μL, or about 0.012 to about 0.018 μg/μL.Higher bead concentrations reduce the time to count the minimum numberof beads (e.g., 50-100 bead events) required to determine the mediansignal for a given reaction vessel. In some embodiments, 200 bead eventsare counted per reaction sample.

In some embodiments, the method further comprises recovering the solidsupports from the incubated sample described above. Particle recoveryand washing can be facilitated by the use of particles that are formedof or contain a magnetically responsive material, i.e., any materialthat responds to a magnetic field. Separation of the solid and liquidphases, either after incubation or after a washing step, is thenachieved by imposing a magnetic field on the reaction vessel in whichthe particles and sample are incubated, causing the particles to adhereto the wall of the vessel and thereby permitting the liquid to beremoved by decantation or aspiration. Magnetically responsive materialsof interest in this invention include paramagnetic materials,ferromagnetic materials, ferrimagnetic materials, and metamagneticmaterials. Examples, include, e.g., iron, nickel, and cobalt, as well asmetal oxides such as Fe₃O₄, BaFe₁₂O₁₉, CoO, NiO, Mn₂O₃, Cr₂O₃, andCoMnP.

Methods of, and instrumentation for, applying and removing a magneticfield as part of an assay are known to those skilled in the art andreported in the literature. Examples of literature reports are Forrestet al., U.S. Pat. No. 4,141,687 (Technicon Instruments Corporation, Feb.27, 1979); Ithakissios, U.S. Pat. No. 4,115,534 (Minnesota Mining andManufacturing Company, Sep. 19, 1978); Vlieger, A. M., et al.,Analytical Biochemistry 205:1-7 (1992); Dudley, Journal of ClinicalImmunoassay 14:77-82 (1991); and Smart, Journal of Clinical Immunoassay15:246-251 (1992).

Magnetically responsive material can be dispersed throughout thepolymer, applied as a coating on the polymer surface or as one of two ormore coatings on the surface, or incorporated or affixed in any othermanner that secures the material in to the particle. The quantity ofmagnetically responsive material in the particle is not critical and canvary over a wide range. The quantity can affect the density of themicroparticle, however, and both the quantity and the particle size canaffect the ease of maintaining the microparticle in suspension forpurposes of achieving maximal contact between the liquid and solid phaseand for facilitating flow cytometry. An excessive quantity ofmagnetically responsive material in the microparticles may produceautofluorescence at a level high enough to interfere with the assayresults. Therefore, in some embodiments, the concentration ofmagnetically responsive material is low enough to minimize anyautofluorescence emanating from the material. With these considerationsin mind, the magnetically responsive material in a particle inaccordance with this invention is, for example, from about 0.05% toabout 75% by weight of the particle as a whole. In some embodiments, theweight percent range is from about 1% to about 50%, e.g., from about 2%to about 25%, e.g., from about 2% to about 8%.

In some embodiments, the binding members are covalently coupled to thesolid support. In some embodiments, the binding member is an antibody tohFXIII, which is covalently linked to the solid support. In someembodiments, the binding members are proteins that are covalentlycoupled to the solid support using carbodiimide-mediated couplingchemistry. Other methods of covalently coupling proteins to a solidsupport are well known in the art. For example, proteins can be coupledto a reactive carboxy group on polystyrene beads. The initial carboxybead can be chemically converted to amino or activated carbon-carbondouble groups. Solid supports can also contain reactive chloroalkylgroups or similar alkylating agents which are capable of reactingdirectly with protein amino residues.

In other embodiments, the binding members are non-covalently coupled tothe solid support. For example, the proteins can be adsorbed to thesurface of the solid support via electrostatic interactions. In someembodiments, the solid support can be coated with streptavidin in orderto capture biotinylated proteins, antigens and antibodies using thestrong avidity of streptavidin for biotin. The streptavidin can becovalently coupled to the solid support using conventional carbodiimideactivation chemistry. Other binding proteins such as Protein G or A canbe used to immobilize antibodies on beads.

In some embodiments, the normalization factor is covalently coupled tothe solid support. In some embodiments, the normalization factor ishFXIII, which is covalently linked to a solid support. For example,hFXIII can be covalently linked to a bead using carbodiimide mediatedcoupling chemistry. In one embodiment, hFXIII is coupled to a uniquebead region. In some embodiments, hFXIII is non-covalently coupled tothe solid support, for example, by adsorption. In some embodiments,hFXIII is indirectly coupled to the solid support, for example, by firstcovalently coupling an antibody to hFXIII to the solid support, and thenincubating the solid support with hFXIII to produce an immunocoupledhFXIII solid support.

In some embodiments, the assay includes a solid support coupled to ananti-human Factor XIII antibody. In one embodiment, the anti-humanFactor XIII antibody is coupled to a bead. In one embodiment, the beadis coupled to a mouse anti-human FXIII monoclonal antibody. Thisembodiment is referred to as a Serum Verification Bead (SVB). The SVB isuseful to detect native human Factor XIII in the sample to verify sampleaddition. Examples of beads coupled to anti-human Factor XIII monoclonalantibodies are described in U.S. Pat. No. 7,141,362, which isincorporated in its entirety by reference herein.

After the solid supports are recovered from the biological sample, forexample, by using the methods described above, the solid supports areincubated with reagents having binding affinity for each of the analytesand reagents having binding affinity for hFXIII. In some embodiments,the reagents are biotinylated conjugates. In some embodiments, thebiotinylated conjugates are biotinylated proteins, peptides, orantibodies. Examples of biotinylated conjugates include:39-PFN-BgG-Biotin for detection of HIV-1/M antibodies; SV2V-biotin fordetection of HIV-2 antibodies; AFR-biotin for detection of HIV-1/Oantibodies; and biotinylated anti-p24 antibodies for detection of p24antigen. In some embodiments, the biotinylated conjugate is abiotinylated antibody that binds to human FXIII.

Methods of biotinylating peptides are well known in the art. Forexample, the most common and versatile method for biotinylation is byreaction of biotin derivatives possessing the N-hydroxysuccinimide estergroup. This chemical functionality allows the formation of stablecovalent amide bonds between biotin and the protein amino group undervery mild reaction conditions (room temperature, neutral to high pH)usually within a short period of time (about 1 hour) to completion.Biotin derivatives are commercially available and the distance andlinkage type between biotin and protein can be tailored to the need ofthe specific target biotin conjugate. Biotin derivatives which containactive hydrazide groups can also be used to link biotin to availablecarbonyl moieties of proteins. These compounds are often used if thereis a need to directly label the protein near its sugar containingdomains. Biotin derivatives which carry N-ethylmaleimide groups orsimilar unsaturated carbon-carbon bonds are used to target proteinthiols and provide another example for site specific labeling ofproteins. Other more specialized biotin derivatives carry chemicalfunctionalities such as aryl azides which can be activatedphotochemically using ultraviolet light. Once photochemically activated,these transient biotin species are allowed to react with various typesof protein residues. See, for example, Richard P. Haugland, Handbook ofFluorescent Probes and Research Chemicals, Chapter 4, Biotins andHaptens, pp, 63-80, Sixth Edition, 1996, which is incorporated byreference herein.

The biotinylated conjugates are incubated under conditions which promotethe binding of the biotinylated conjugates to hFXIII and the analytes.Suitable conditions include phosphate buffer with NaCl, proteinstabilizers, blockers, and preservatives. The pH can be in the range ofabout 7.0-7.4.

In some embodiments, after the solid supports are incubated with thebiotinylated conjugates, excess conjugate is removed by washing and thesolid supports are incubated with a labeled binding moiety. In someembodiments, the labeled binding moiety is selected from streptavidinand avidin. In some embodiments, the labels are fluorophores, many ofwhich are reported in the literature and thus known to those skilled inthe art, and many of which are readily available from commercialsuppliers to the biotechnology industry. Literature sources forfluorophores include Cardullo et al., Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988); Dexter, D. L., J. of Chemical Physics 21: 836-850(1953); Hochstrasser et al., Biophysical Chemistry 45: 133-141 (1992);Selvin, P., Methods in Enzymology 246: 300-334 (1995); Steinberg, I.,Ann. Rev. Biochem., 40: 83-114 (1971); Stryer, L., Ann. Rev. Biochem.47: 819-846 (1978); Wang et al., Tetrahedron Letters 31: 6493-6496(1990); and Wang et al., Anal. Chem. 67: 1197-1203 (1995).

The following are examples of fluorophores that can be used as labels:

-   -   4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid    -   acridine    -   acridine isothiocyanate    -   5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)    -   4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate    -   N-(4-anilino-1-naphthyl)maleimide    -   anthranilamide    -   BODIPY    -   Brilliant Yellow    -   coumarin    -   7-amino-4-methylcoumarin (AMC, Coumarin 120)    -   7-amino-4-trifluoromethylcoumarin (Coumaran 151)    -   cyanine dyes    -   cyanosine    -   4′,6-diaminidino-2-phenylindole (DAPI)    -   5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red)    -   7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin    -   diethylenetriamine pentaacetate    -   4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid    -   4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid    -   5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,        dansylchloride)    -   4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)    -   4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC)    -   eosin    -   eosin isothiocyanate    -   erythrosin B    -   erythrosin isothiocyanate    -   ethidium    -   5-carboxyfluorescein (FAM)    -   5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)    -   2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE)    -   fluorescein    -   fluorescein isothiocyanate    -   fluorescamine    -   IR144    -   IR1446    -   Malachite Green isothiocyanate    -   4-methylumbelliferone    -   ortho cresolphthalein    -   nitrotyrosine    -   pararosaniline    -   Phenol Red    -   phycoerythrin (including but not limited to B and R types)    -   o-phthaldialdehyde    -   pyrene    -   pyrene butyrate    -   succinimidyl 1-pyrene butyrate    -   quantum dots    -   Reactive Red 4 (Cibacron Brilliant Red 3B-A)    -   6-carboxy-X-rhodamine (ROX)    -   6-carboxyrhodamine (R6G)    -   lissamine rhodamine B sulfonyl chloride rhodamine    -   rhodamine B    -   rhodamine 123    -   rhodamine X isothiocyanate    -   sulforhodamine B    -   sulforhodamine 101    -   sulfonyl chloride derivative of sulforhodamine 101 (Texas Red)    -   N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA)    -   tetramethyl rhodamine    -   tetramethyl rhodamine isothiocyanate (TRITC)    -   riboflavin    -   rosolic acid    -   lanthanide chelate derivatives

A prominent group of fluorophores for immunoassays are fluorescein,fluorescein isothiocyanate, phycoerythrin, rhodamine B, and Texas Red(sulfonyl chloride derivative of sulforhodamine 101). Phycoerythrin isparticularly prominent. Thus, in one embodiment, the labeled bindingmoiety is streptavidin conjugated with phycoerythrin (PE). Any of thefluorophores in the list preceding this paragraph can be attached tobinding members by conventional covalent bonding, using appropriatefunctional groups on the fluorophores and on the binding moieties. Therecognition of such groups and the reactions to form the linkages willbe readily apparent to those skilled in the art.

Other labels that can be used in place of the fluorophores areradioactive labels and enzyme labels. These are likewise known in theart.

After the solid supports are incubated with the labeled binding member,the solid supports are recovered using the recovery methods describedabove, and the amount of label that is bound to the supports isdetected. In some embodiments, the detecting step is accomplished bymeasuring the amount of fluorescence signal produced by the fluorescentlabels described above.

In some embodiments, the fluorescence signal is detected by flowcytometry. Flow cytometry is used for bead size classification(forward/side scatter) with a dual laser system to classify bead dyedregions and then quantitate the fluorescent signal from each region.Other multiplex methods for identification of solid phase populationsand the associated population signal are known to those of skill in theart. Examples include Luminex Magpix® technology using stop-flowimmobilization of beads followed by identification and quantitationusing LED-CCD imaging (see the internet atluminexcorp.com/Products/Instruments/MAGPIX/). Another example isIlumina Veracode™ multiplex technology which uses cylindrical glassmicrobeads having high-density codes as the solid phase and a grooveplate detection method (see the internet atillumina.com/systems/beadxpress/technology.ilmn). Other examples ofmultiplex technology include chip based assays such as Randox BioChipArray Technology assays (see the internet at randox.com/Biochip%20Immunoassays.php).

In some embodiments, the fluorescent signal is generated usingLanthanide chelate reagents and is detected using time-resolvedfluorescence. Lanthanide chelates are unique in that their fluorescencedecays exhibit longer lifetimes and larger “Stoke's Shifts”. The use ofLanthanide chelates allows higher sensitivities for immunoassay analytedetection. The chelates can be custom synthesized and linked to proteinsand antibodies via analogous chemical conjugation techniques forconventional fluorophores like Fluorescein, Rhodamine, Phycoerythrin andothers. The metal associated with the chelate that is most commonly usedis Europium. A commercial system based on this technology is the DELFIA®(Dissociation-Enhanced Lanthanide Fluorescent Immunoassay) TRF (timeresolved fluorescence) assay system (Perkin Elmer, Waltham. MA).

In some embodiments, the amount of label detected is correlated with thedifferentiation parameters described herein to obtain values that areindividually representative of the levels of the analytes in thebiological sample. In some embodiments, the values representative of thelevels of the analytes are normalized to the values representative ofthe levels of the normalization factor. For example, in someembodiments, the values are normalized by dividing the valuerepresentative of the level of the analyte by the value representativeof the level of the normalization factor (e.g., the ratio of the analytesignal/human FXIII signal), and multiplying the result by a constant toproduce a normalized value. The normalized value (also referred to asthe normalized signal) can be used to correct for variations in sampleprocessing and biological matrix effects during performance of themethod. In some embodiments, the normalized values significantly reducethe coefficient of variation (percent relative standard deviation)between different replicate assays using the same biological sample.

Signal normalization provides improved assay precision for samples,controls and calibrators used in the methods. Signal normalization alsoprovides improved signal stability over the lifetime of the reagentsused in the methods. It will be understood that the normalization factorcan also be used to normalize values in assays for detecting otherbiological conditions in a subject, such as viral infections in additionto HIV, and/or other pathological conditions, such as acute or chronicdisease.

II. Functional Groups

Coating of the particle surface with the appropriate assay reagent canbe achieved by electrostatic attraction, specific affinity interaction,hydrophobic interaction, or covalent bonding. The polymer can bederivatized with functional groups for covalent attachment of the assayreagents by conventional means, notably by the use of monomers thatcontain the functional groups, such monomers serving either as the solemonomer or as a co-monomer. Examples of suitable functional groups areamine groups (—NH₂), ammonium groups (—NH₃ ⁺ or —NR₃ ⁺), hydroxyl groups(—OH), carboxylic acid groups (—COOH), and isocyanate groups (—NCO).Useful monomers for introducing carboxylic acid groups into polyolefins,for example, are acrylic acid and methacrylic acid.

Linking groups can be used as a means of increasing the density ofreactive groups on the particle surface and also as a means ofdecreasing steric hindrance. Linking groups can also be used as a meansof securing coating materials to the particle surfaces. Certain linkinggroups are monofunctional linkers comprising a reactive group as well asmultifunctional crosslinkers comprising two or more reactive groupscapable of forming a bond with two or more different functional targets(e.g., peptides, proteins, macromolecules, semiconductor nanocrystals,or substrate). In some embodiments, the multifunctional crosslinkers areheterobifunctional crosslinkers comprising two different reactivegroups. Examples of suitable reactive groups are thiol (—SH),carboxylate (—COOR), carboxyl (—COOH), carbonyl (—C(O)—), amine (NH₂),hydroxyl (—OH), aldehyde (—CHO), hydroxyl (—OH), active hydrogen, ester,phosphate (—PO₃), and photoreactive moieties. Examples of amine reactivegroups are isothiocyanates, isocyanates, acyl azides, NHS esters,sulfonyl chlorides, aldehydes and glyoxals, epoxides and oxiranes,carbonates, arylating agents, imidoesters, carbodiimides, andanhydrides. Examples of thiol-reactive groups are haloacetyl and alkylhalide derivates, maleimides, aziridines, acryloyl derivatives,arylating agents, and thiol-disulfides exchange reagents. Examples ofcarboxylate reactive groups are diazoalkanes and diazoacetyl compounds,such as carbonyldiimidazoles and carbodiimides. Examples of hydroxylreactive groups are epoxides and oxiranes, carbonyldiimidazole,oxidation with periodate, N,N′-disuccinimidyl carbonate orN-hydroxylsuccimidyl chloroformate, enzymatic oxidation, alkyl halogens,and isocyanates. Examples of aldehyde and ketone reactive groups arehydrazine derivatives for Schiff base formation or reduction amination.Examples of active hydrogen reactive groups are diazonium derivativesfor Mannich condensation and iodination reactions. Examples ofphotoreactive groups are aryl azides and halogenated aryl azides,benzophenones, diazo compounds, and diazirine derivatives.

Other suitable reactive groups and classes of reactions useful inpracticing the present invention are generally those that are well knownin the art of bioconjugate chemistry. Currently favored classes ofreactions available with reactive chelates are those which proceed underrelatively mild conditions. These include, but are not limited to,nucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, Advanced OrganicChemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney etal., Modification Of Proteins; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

In some embodiments, the functional group is a heterobifunctionalcrosslinker comprising two different reactive groups that containheterocyclic rings that can interact with peptides and proteins. Forexample, heterobifunctional crosslinkers such asN-[γ-maleimidobutyryloxy]succinimide ester (GMBS) or succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) comprise an aminereactive group and a thiol-reactive group that can interact with aminoand thiol groups within peptides or proteins. Additional combinations ofreactive groups suitable for heterobifunctional crosslinkers include,for example, carbonyl and sulfhydryl reactive groups; amine andphotoreactive groups; sulfhydryl and photoreactive groups; carbonyl andphotoreactive groups; carboxylate and photoreactive groups; and arginineand photoreactive groups. Examples of suitable useful linking groups arepolylysine, polyaspartic acid, polyglutamic acid and polyarginine.N-hydroxysuccinimide (NHS), CMC1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC),N-Hydroxybenzotriazole (HOBt), and/or other crosslinking agents may beused.

Particles formed by conventional emulsion polymerization techniques froma wide variety of starting monomers are favorable in many cases sincethey exhibit at most a low level of autofluorescence. Conversely,particles that have been modified to increase their porosity and hencetheir surface area, i.e., those particles that are referred to in theliterature as “macroporous” particles, tend to exhibit highautofluorescence and are often less desirable. Autofluorescenceincreases with increasing size and increasing amounts of divinylbenzenemonomer.

Multiplexing, i.e., the performance of simultaneous assays for allanalytes in a given panel, can be performed with the use of solidsupports by utilization of differentiation parameters, as mentionedabove and described below.

III. Differentiation Parameters

One example of a differentiation parameter is the particle diameter,where the solid supports are particles divided into groups withnonoverlapping diameter subranges. The widths of the diameter subrangesand the spacing between mean diameters of adjacent subranges in theseembodiments are selected to permit differentiation of the subranges byflow cytometry, and such selection will be readily apparent to thoseskilled in the use of and instrumentation for flow cytometry. In thisspecification, the term “mean diameter” refers to a number averagediameter. In some embodiments, the subrange width is about ±5% CV orless of the mean diameter, where “CV” stands for “coefficient ofvariation” and is defined as the standard deviation of the particlediameter divided by the mean particle diameter, times 100 percent. Theminimum spacing between mean diameters among the various subranges canvary depending on the microparticle size distribution, the ease ofsegregating microparticles by size for purposes of attaching differentassay reagents, and the type and sensitivity of the flow cytometryequipment. In some embodiments, best results will be achieved when themean diameters of different subranges are spaced apart by at least about6% of the mean diameter of one of the subranges, e.g., at least about 8%of the mean diameter of one of the subranges, e.g., at least about 10%of the mean diameter of one of the subranges. In some embodiments, thestandard deviation of the particle diameters within each subrange isless than one third of the separation of the mean diameters of adjacentsubranges.

Another example of a differentiation parameter that can be used todistinguish among different groups of particles is fluorescence.Differentiation by fluorescence is accomplished by incorporating one ormore fluorescent materials in the particles, the fluorescent materialshaving different fluorescent emission spectra and being distinguishableon this basis. Differentiation can be achieved by using fluorescentmaterials that have different fluorescence intensities or that emitfluorescence at different wavelengths, or by varying the amount offluorescent material incorporated. Differentiation by fluorophores canalso be achieved by using combinations of fluorophores for each particlesubgroup. For example, the particle can be made to contain a redfluorochrome such as Cy5 together with a far-red fluorochrome such asCy5.5, at different relative amounts for different subgroups. Additionalfluorochromes can be used to further expand the system. Eachmicroparticle can thus contain a plurality of fluorescent dyes atvarying wavelengths.

By using fluorescence emissions at different wavelengths, the wavelengthdifference can be used to distinguish the particle groups from eachother, while also distinguishing the labels in the labeled anti-humanantibodies from the labels that differentiate one particle group fromanother. An example of a fluorescent substance that can be used as ameans of distinguishing particle groups is fluorescein and an example ofa substance that can be used for the assay detection is phycoerythrin.In the use of this example, different particle groups can be dyed withdiffering concentrations of fluorescein to distinguish them from eachother, while phycoerythrin is used as the label on the various labeledbinding members used in the assay.

Another example of a differentiation parameter that can be used todistinguish among the various groups of particles is light scatter. Sideangle light scatter varies with particle size, granularity, absorbanceand surface roughness, while forward angle light scatter is mainlyaffected by size and refractive index. Varying any of these qualitiescan result in light scatter differences that can serve as a means ofdistinguishing the various groups.

Still another example of a differentiation parameter is absorbance. Whenlight is applied to particles, the absorbance of the light by theparticles is indicated mostly by a change in the strength of thelaterally (side-angle) scattered light while the strength of theforward-scattered light is relatively unaffected. Consequently, thedifference in absorbance between various colored dyes associated withthe particles is determined by observing differences in the strength ofthe laterally scattered light.

A still further example of a differentiation parameter is the number ofparticles in each group. When the number of particles in each group isvaried in a known way, the count of particles having various assayresponses can be associated with a particular assay by the number ofparticles having each response.

As the above examples illustrate, a wide array of parameters orcharacteristics can be used as differentiation parameters to distinguishthe particles of one group from those of another. The differentiationparameters may arise from particle size, from particle composition, fromparticle physical characteristics that affect light scattering, fromexcitable fluorescent dyes or colored dyes that impart differentemission spectra and/or scattering characteristics to the particles, orfrom different concentrations of one or more fluorescent dyes. When thedistinguishable particle parameter is a fluorescent dye or color, it canbe coated on the surface of the particle, embedded in the particle, orbound to the molecules of the particle material. Thus, fluorescentparticles can be manufactured by combining the polymer material with thefluorescent dye, or by impregnating the particle with the dye. Particleswith dyes already incorporated and thereby suitable for use in thepresent invention are commercially available, from suppliers such asSpherotech, Inc. (Libertyville, Ill., USA) and Molecular Probes, Inc.(Eugene, Oreg., USA).

When particles are used, particularly microparticles, the use of flowcytometry is a convenient way of sorting the particles by thedifferentiation parameter, and also in many cases of determining whethera label has been attached to the particle through the assay componentsas a result of the assay reaction.

Methods of, and instrumentation for, flow cytometry are known in theart, and can be used in the practice of the present invention. Flowcytometry in general resides in the passage of a suspension of particles(or cells) in as a stream through a light beam and coupled toelectro-optical sensors, in such a manner that only one particle at atime passes the region of the sensors. As each particle passes thisregion, the light beam is perturbed by the presence of the particle, andthe resulting scattered and fluoresced light are detected. The opticalsignals are used by the instrumentation to identify the subgroup towhich each particle belongs, along with the presence and amount oflabel, so that individual assay results are achieved. Descriptions ofinstrumentation and methods for flow cytometry are found in theliterature. Examples are McHugh, “Flow Microsphere Immunoassay for theQuantitative and Simultaneous Detection of Multiple Soluble Analytes,”Methods in Cell Biology 42, Part B (Academic Press, 1994); McHugh etal., “Microsphere-Based Fluorescence Immunoassays Using Flow CytometryInstrumentation,” Clinical Flow Cytometry, Bauer, K. D., et al., eds.(Baltimore, Md., USA: Williams and Williams, 1993), pp. 535-544; Lindmoet al., “Immunometric Assay Using Mixtures of Two Particle Types ofDifferent Affinity,” J. Immunol. Meth. 126: 183-189 (1990); McHugh,“Flow Cytometry and the Application of Microsphere-Based FluorescenceImmunoassays,” Immunochemica 5: 116 (1991); Horan et al., “Fluid PhaseParticle Fluorescence Analysis Rheumatoid Factor Specificity Evaluatedby Laser Flow Cytophotometry,” Immunoassays in the Clinical Laboratory,185-189 (Liss 1979); Wilson et al., “A New Microsphere-BasedImmunofluorescence Assay Using Flow Cytometry,” J. Immunol. Meth. 107:225-230 (1988); Fulwyler et al., “Flow Microsphere Immunoassay for theQuantitative and Simultaneous Detection of Multiple Soluble Analytes,”Meth. Cell Biol. 33: 613-629 (1990); Coulter Electronics Inc., UnitedKingdom Patent No. 1,561,042 (published Feb. 13, 1980); and Steinkamp etal., Review of Scientific Instruments 44(9): 1301-1310 (1973).

The methods of the present invention, and the kits of the presentinvention that contain materials for use in practicing the methods,allow for the simultaneous detection and optionally quantification ofthe various analytes in a biological sample. The presence of theseanalytes or panel of analytes can be an indication of the presence,absence, or stage of a biological condition in the subject from whom thesample was taken. In some embodiments, the detection and/orquantification of some or all of the various analytes in a sample isused to provide a prognosis or to assess the efficacy of apharmaceutical treatment (e.g., an anti-viral drug). Diagnosis,prognosis, or assessing pharmaceutical efficacy can be achieved forexample by correlating the amounts of certain analytes in the samplewith known amounts associated with healthy individuals, diseasedindividuals, or both. In some embodiments, the diagnosis or prognosiscan be used to recommend a course of treatment for the subject.

In another embodiment, the normalization factor can be used to normalizesignal values as assay reagents degrade over time. For example, the SNBsignal (like the assay signal) is sensitive to concentrations of boththe biotinylated conjugate and the streptavidin-phycoerythrin (SA-PE)reagent. As these reagents degrade on storage or under elevatedtemperature associated with shipping stress, a loss of signal istypically observed for both the SNB and the assay bead. Using SNBnormalization maintains the assay response stability despite theabsolute loss of assay signal. This has advantages for assay sensitivityrelated to calibration curve slope.

EXAMPLES Example 1

This example demonstrates that a hFXIII bead included in an HIVdetection assay can be used to detect unexpected and unacceptablevariations in assay signal.

Methods:

All incubations are at 37° C. 25 μL of bead reagent containing beads fordetection of HIV analytes, hFXIII, SVB, and ISB, and 25 μL sample areincubated for 28.5 min, then washed. 50 μL of Conjugate 1 reagentcontaining biotinylated conjugates is added to the beads and incubated10 min, then washed. 25 μL Conjugate 2 containing concentrated SA-PE and25 μL wash fluid are added to the beads and incubated 10 min. Afterwashing, beads are resuspended in 50 uL wash fluid and sent to thedetector. Beads are in a buffer containing triethanolamine and CHAPS(3[(3-Cholamidopropyl)dimethylammonio]-propanesulfonic acid), proteinstabilizers, blockers, and preservatives. Conjugate 1 and Conjugate 2are in phosphate buffer with NaCl, protein stabilizers, blockers, andpreservatives. All buffers are pH 7-7.4. The starting concentration ofStreptavidin-PE was 4-8 μg/mL in the reagent. During reaction vesselprocessing, 25 μL of Streptavidin-PE reagent was mixed with 25 uL ofdiluent for a final working concentration of 2-4 μg/mL.

Biological samples containing HIV antigen (p24 antigen) were incubatedwith magnetic beads covalently coupled to anti-p24 antibodies and withmagnetic beads covalently coupled to hFXIII (referred to herein as ahFXIII bead or signal normalization bead (SNB)). Following theincubation step, the beads were separated from the sample, washed, andincubated with biotinylated antibodies that specifically bind p24antigen and hFXIII (mouse anti-human FXIII-biotin). The concentration ofbiotin conjugated anti-p24 antibody was 5.0 μg/mL. The concentration ofbiotin conjugated anti-p24 antibody was 1.5 μg/mL. Excess conjugate wasremoved by washing, and streptavidin-PE conjugate was added to thebeads. Following incubation with streptavidin-PE, the beads were washedand the amount of fluorescent signal was detected. Twelve (12) replicateassays were performed.

As shown in FIG. 1, the amount of HIV antigen signal and the amount ofhFXIII signal were positively correlated for each replicate. Further,the normalized signal (antigen signal/hFXIII signal multiplied by 150)showed much less variability than the antigen signal between replicates.As shown in Table 2, the coefficient of variation (percent relativestandard deviation, CV %) was much less using the normalized signal thaneither the antigen signal or hFXIII signal. Further, the hFXIII signalcan be used to detect an invalid assay result, such as when the hFXIIIsignal falls below a preset limit (e.g., a signal for hFXIII below 250).

TABLE 2 Analysis of 12 Replicates of Antigen Positive Sample ReplicateID Antigen hFXIII Normalized 1 239.0 338.5 105.9 2 248.0 359.0 103.6 3257.0 351.5 109.7 4 263.0 337.5 116.9 5 248.0 348.0 106.9 6 145.0 200.0108.8 7 253.0 348.5 108.9 8 270.0 424.0 95.5 9 238.0 320.5 111.4 10 250.0 360.5 104.0 11  247.0 344.5 107.5 12  267.0 362.0 110.6 Mean 243.8341.2 107.5 Std Dev 32.7 51.0 5.2 CV % 13.4 14.9 4.8

This example demonstrates that using a hFXIII bead to normalize analytevalues produced improved assay precision. This example furtherdemonstrates that the hFXIII signal normalization bead can be used as acontrol bead to detect an invalid assay.

Example 2

This example demonstrates that using a hFXIII bead significantlyimproves the CV of the normalized signal when performing replicates of amultiplex control sample.

A multiplex control sample containing human polyclonal antibodies forHIV-1 and HIV-2 as well as rabbit monoclonal antibodies representingHIV-O was incubated with magnetic beads covalently coupled to antigensthat specifically bind to the antibodies (target analyte beads). TheHIV-1 detection bead was ligated to GP-160. The HIV-2 detection bead wasligated to SPOH. The HIV-O detection bead was ligated to AFR. Thecontrol sample was also incubated with hFXIII beads. The sample wasprocessed as described in Example 1. Five (5) replicates of the assaywere performed.

As shown in FIG. 2, the HIV-1 control signal was positively correlatedwith the hFXIII signal in each replicate. The normalized signal showedmuch less variation between replicates. As shown in Table 3, the CV % ofthe normalized signal was significantly lower that the CV % of eitherthe HIV-1 signal or the hFXIII signal.

TABLE 3 Analysis of five replicates of HIV-1 antibody positive controlsample. HIV-1 Normalized HIV-1 hFXIII Signal 1 L03-QC 217 248 87.5 2L03-QC 203 221 91.9 3 L03-QC 323 355 90.8 4 L03-QC 300 323 92.9 5 L03-QC319 335 95.2 Mean 272 296 92 Std Dev 57.7 58.4 2.8 CV % 21.2 19.7 3.1

Similarly, FIG. 3 shows that the HIV-2 control signal was positivelycorrelated with the hFXIII signal in each replicate. The normalizedsignal showed much less variation between replicates. Table 4 shows thatthe CV % of the normalized signal was significantly lower that the CV %of either the HIV-2 signal or the hFXIII signal.

TABLE 4 Analysis of five replicates of HIV-2 antibody positive controlsample. HIV-2 Normalized HIV-2 hFXIII Signal 1 L03-QC 318 248 128.2 2L03-QC 282 221 127.6 3 L03-QC 451 355 127.0 4 L03-QC 426 323 132.1 5L03-QC 426 335 127.2 Mean 381 296 128 Std Dev 75.4 58.4 2.1 CV % 19.819.7 1.6

Similar results were obtained with the HIV-O control sample. FIG. 4shows that the HIV-O control signal was positively correlated with thehFXIII signal in each replicate. The normalized signal showed much lessvariation between replicates. Table 5 shows that the CV % of thenormalized signal was significantly lower that the CV % of either theHIV-O signal or the hFXIII signal.

TABLE 5 Analysis of five replicates of HIV-O antibody positive controlsample. HIV-O Normalized HIV-O hFXIII Signal 1 L03-QC 257 248 103.6 2L03-QC 222 221 100.5 3 L03-QC 339 355 95.5 4 L03-QC 328 323 101.7 5L03-QC 326 335 97.3 Mean 294 296 100 Std Dev 51.9 58.4 3.3 CV % 17.619.7 3.3

Example 3

This example describes factors that influence the final fluorescentsignal generated on the beads.

During development of the above assays, it was observed that replicateanalysis of samples such as calibrators, controls or patient specimens(serum or plasma) could occasionally produce variable fluorescentsignals on the assay beads resulting in high coefficients of variation.In some cases, the signal variation could be as high as 50% or more andwas sufficient to produce a response classified as positive for somereplicates but negative for others.

Numerous factors associated with assay chemistry as well as manual orautomated sample processing are responsible for the magnitude of thefinal fluorescent signal generated on the beads. Some sources of signalfluctuation between replicates can be related to assay variations listedin Table 6.

TABLE 6 Factors that introduce signal fluctuation between replicates.Step-1 Bead - Sample variations sample dispense volume bead dispensevolume bead-sample incubation time bead-sample mixing efficiencybead-sample incubation temperature bead-sample washing efficiency toremove bulk sample after analyte binding to beads Volume of residualwash reagent after bead-sample wash steps Step-2 Bead - Conjugatevariations Biotin conjugate concentration Biotin conjugate volumebead-conjugate incubation time bead-conjugate mixing efficiencybead-conjugate incubation temperature bead-conjugate washing efficiencyto remove bulk conjugate after conjugate binding to beads Volume ofresidual wash reagent after bead-conjugate wash steps Step-3 Bead - PEvariations PE dispense volume bead-PE incubation time bead-PE mixingefficiency bead-PE incubation temperature bead-PE washing efficiency toremove bulk PE after analyte binding to beads and prior to detectiontime from bead-PE binding to fluorescent detection Detector response(drift) due to temperature or voltage over time

Since hFXIII is covalently coupled to beads, it is not responsive tovariations in the analyte-bead binding steps described in Step-1. It ishowever responsive to the final process shown in Step-1 (volume ofresidual wash reagent after bead-sample wash steps) as this affects thefinal concentration of biotin reagent added in Step-2. The bead isresponsive to other processes listed in Steps 2 and Step 3.

Example 4

This example demonstrates that normalization of signal values using thehFXIII signal normalization bead reduces variation between replicatescompared to an internal standard bead coated with tetramethylcadaverinerhodamine (TMRC).

Since the fluorecence detector response may drift over time, some manualor automated assays typically include an Internal Standard Bead (ISB)coated with a fixed amount of TMRC, thus producing a standard amount ofsignal. Assay signals can be normalized to the ISB signal to compensatefor detector variation. However, since ISB does not participate in theimmunoassay, it does not respond to potential process variationsdescribed in Table 6 other than detector drift. For a series ofreplicates, no improvement in assay precision was observed whennormalizing signals to ISB as shown below in Table 7 for the HIV-1 datadescribed above in Example 2.

TABLE 7 Comparison of the normalized signal using an ISB and hFXIII SNB.HIV-1 Raw hF13 Raw ISB Raw HIV-1/ISB *8000 HIV-1/hF13*100 ReplicateAccession Signal Signal Signal Normalized Signal Normalized Signal 1L03-QC 217 248 8201 212 87.5 2 L03-QC 203 221 8137 200 91.9 3 L03-QC 323355 8147 317 90.8 4 L03-QC 300 323 7935 302 92.9 5 L03-QC 319 335 8027.5318 95.2 Mean 272 296 8090 270 92 Std Dev 57.7 58.4 106.9 58.9 2.8 CV %21.2 19.7 1.3 21.8 3.1

Example 5

This example demonstrates that signal variation is affected by residualbiotin conjugate in the reaction vessel prior to addition ofstreptavidin-PE.

The inventors observed that if biotin conjugate is not efficientlyremoved during wash phase of Step 2 in Table 6 (e.g., due to instrumentwash-aspirate errors), signal is significantly reduced due to theinteraction of residual aqueous biotin conjugate with PE. The result isan effective reduction in PE concentration. This type of error ismodeled below for HIV-O antibody detection in antibody calibrator:

Four test methods (labeled HIV, HIV2, HIV3, HIV4 on the X axis) werecreated in which various amounts of biotin conjugate were purposefullyleft in the reaction vessel (RV) prior to the addition of thefluorescent reporter (PE). FIG. 5 shows the signal response for the testmethods.

HIV represents the standard method with full RV washing protocol (8replicates shown). HIV-2 and HIV-3 show the complete loss of signal when10 uL or 5 uL, respectively, of residual biotin conjugate arepurposefully added to the RV prior to the addition of PE reagent. HIV-4shows the effect of poor incomplete washing of the RV prior to theaddition of the PE reagent. The ISB signal remained constant (data notshown).

In separate experiments exploring the use of an alternate internalstandard bead coupled directly with biotin, it was found that if thebiotin bead was added in sufficient quantity (ie solid phase biotin),the assay signals will also drop. The ISB does not detect this drop inassay signals, but since it appears to be an effect on free PEconcentration, the SNB tracks the assay signal producing good precision.

Likewise, if biotin conjugate dispense volume is lower than expected dueto instrument pipetting error, or if residual wash volume is higher thanexpected due to instrument wash aspirate error, the final conjugateconcentration in the reaction mixture is reduced resulting in lowerassay signal and lower hFXIII signal while the ISB signal remainsconstant.

In summary, this example demonstrates that the hFXIII SNB can be used tonormalize the assay signal when residual biotin conjugate remains in thereaction vessel prior to the addition of labeled streptavidin.

Example 6

This example demonstrates that biological matrix effects can producevariable recovery of p24 antigen signal, and that this variability issignificantly reduced when using the hFXIII bead for normalization.

A human donor provided blood specimens drawn into 9 different types ofcollection tubes. The glass or plastic collection tubes containedvarious anticoagulants for production of serum or plasma. Serum wasobtained from tubes without anticoagulant. Some tubes contain serumseparator (SST) or plasma separator (PST) barriers for separation ofcells from serum or plasma. The tube types are described below:

Specimen Type Anticoagulant Glass Plastic Serum None x x Serum SST Nonex x Plasma K2-EDTA x Plasma Sodium Citrate x Plasma Sodium Heparin xPlasma Lithiujm Heparin x Plasma PST Lithium Heparin x

Each serum or plasma matrix was supplemented with an identicalconcentration of HIV p24 antigen and tested with 5 replicates percondition according to the method in Example 1.

In this example all replicates and sample matrix types were expected toproduce the same response for Ag RFI. However, as shown in Table 8 andFIG. 6, there was substantial variation between the different matrixtypes. The % CV of the p24 antigen signal for all replicates and sampletypes was 10.1%. In contrast, when the data was normalized using hFXIII,the variability was significantly reduced. As shown in Table 8 and FIG.7, the % CV for the normalized response (p24 signal/hFXIII signal) wasimproved to 4.6%.

TABLE 8 Detection of HIV p24 antigen RF1 in serum and plasma samplescollected in different types of collection tubes and treated withdifferent anticoagulants and normalized to signal from hFXIII. Ag RFIhFXIII hFXIII Normalized Normalized TMRC Ag RFI (% of RFI RFI (% of (Ag(% of TMRC RFI (% Description ID Replicate Signal mean) Signal mean)RFI/hFXIII) mean) RFI of mean) Serum-Plastic AgSPD1 1 183 93 2001 92 137102 9280 96 Serum-Plastic AgSPD1 2 183 93 1977 90 139 103 9500 98Serum-Plastic AgSPD1 3 175 89 1976 90 133 99 9604 99 Serum-PlasticAgSPD1 4 182 93 1976 90 138 103 9521 98 Serum-Plastic AgSPD1 5 191 972105 96 136 101 9495 98 Serum-Glass AgSGD1 1 194 99 2182 100 133 99 9729101 Serum-Glass AgSGD1 2 201 102 2130 97 141 105 9284 96 Serum-GlassAgSGD1 3 188 96 2198 101 128 95 9886 102 Serum-Glass AgSGD1 4 195 992091 96 140 104 9591 99 Serum-Glass AgSGD1 5 192 98 2157 99 133 99 10016104 Serum-SST-Plastic AgSSTPD1 1 192 98 2227 102 129 96 9498 98Serum-SST-Plastic AgSSTPD1 2 202 103 2233 102 136 101 9758 101Serum-SST-Plastic AgSSTPD1 3 195 99 2112 97 139 103 9892 102Serum-SST-Plastic AgSSTPD1 4 182 93 2012 92 135 101 9390 97Serum-SST-Plastic AgSSTPD1 5 187 95 2054 94 137 102 9724 100Serum-SST-Glass AgSSTGD1 1 169 86 1937 89 130 97 9588 99 Serum-SST-GlassAgSSTGD1 2 176 89 2049 94 128 96 9523 98 Serum-SST-Glass AgSSTGD1 3 17388 1999 91 130 97 9470 98 Serum-SST-Glass AgSSTGD1 4 174 89 1999 91 13197 9640 100 Serum-SST-Glass AgSSTGD1 5 173 88 1956 89 133 99 9510 98Plasma-K2 EDTA AgK2D1 1 167 85 1937 89 129 96 9582 99 Plasma-K2 EDTAAgK2D1 2 169 86 1996 91 127 94 9523 98 Plasma-K2 EDTA AgK2D1 3 164 841947 89 126 94 10026 104 Plasma-K2 EDTA AgK2D1 4 168 86 1986 91 127 949594 99 Plasma-K2 EDTA AgK2D1 5 174 89 2050 94 127 95 10023 104Plasma-Citrate AgCTD1 1 200 102 2269 104 132 98 9873 102 Plasma-CitrateAgCTD1 2 209 107 2278 104 138 102 9913 102 Plasma-Citrate AgCTD1 3 202103 2342 107 129 96 10146 105 Plasma-Citrate AgCTD1 4 203 103 2429 111125 93 9578 99 Plasma-Citrate AgCTD1 5 204 104 2284 104 134 100 9631 100Plasma NaHeparin AgNAHD1 1 202 103 2364 108 128 95 10032 104 PlasmaNaHeparin AgNAHD1 2 205 105 2456 112 125 93 9700 100 Plasma NaHeparinAgNAHD1 3 214 109 2380 109 135 100 10073 104 Plasma NaHeparin AgNAHD1 4205 105 2371 108 130 96 9577 99 Plasma NaHeparin AgNAHD1 5 206 105 2323106 133 99 9654 100 Plasma LiHeparin AgLIHD1 1 234 119 2333 107 150 1129802 101 Plasma LiHeparin AgLIHD1 2 229 117 2480 113 139 103 9779 101Plasma LiHeparin AgLIHD1 3 219 112 2482 113 132 98 9796 101 PlasmaLiHeparin AgLIHD1 4 239 122 2504 115 143 106 9545 99 Plasma LiHeparinAgLIHD1 5 231 118 2338 107 148 110 9530 98 Plasma LiHeparin AgPSTD1 1213 109 2253 103 142 105 9839 102 PST Plasma LiHeparin AgPSTD1 2 222 1132336 107 142 106 9458 98 PST Plasma LiHeparin AgPSTD1 3 220 112 2266 104145 108 9827 102 PST Plasma LiHeparin AgPSTD1 4 210 107 2313 106 136 1019426 97 PST Plasma LiHeparin AgPSTD1 5 218 111 2305 105 142 105 9656 100PST Ag RFI hFXIII RFI hFXIII RFI Normalized Normalized TMRC RFI Ag RFISignal (% of mean) Signal (% of mean) (RFI/hFXIII) (% of mean) TMRC RFI(% of mean) Signal 196.2 100.0 2186.3 100.0 134.5 100.0 9677.2 100.0Stdev 19.8 10.1 174.9 8.0 6.2 4.6 212.6 2.2 % CV 10.1 10.1 8.0 8.0 4.64.6 2.2 2.2

This example shows that using hFXIII to normalize the signalsignificantly reduced the variability associated with different matrixeffects.

In the claims appended hereto, the term “a” or “an” is intended to mean“one or more.” The term “comprise” and variations thereof such as“comprises” and “comprising,” when preceding the recitation of a step oran element, are intended to mean that the addition of further steps orelements is optional and not excluded. All patents, patent applications,and other published reference materials cited in this specification arehereby incorporated herein by reference in their entirety. Anydiscrepancy between any reference material cited herein or any prior artin general and an explicit teaching of this specification is intended tobe resolved in favor of the teaching in this specification. Thisincludes any discrepancy between an art-understood definition of a wordor phrase and a definition explicitly provided in this specification ofthe same word or phrase.

What is claimed is:
 1. A method for normalizing results, the methodcomprising: (a) contacting a biological sample with one or more solidsupports having binding members immobilized thereon that specificallybind an analyte in the biological sample, under conditions promotingbinding of the analyte to the binding members, the solid supports beingdivided into subpopulations that are differentiable from each other by adifferentiation parameter comprising a characteristic that isindependent of the binding members immobilized on the solid supports,the binding members immobilized on each subpopulation binding to oneanalyte in the sample; (b) contacting the biological sample with a solidsupport having human blood coagulation Factor XIII (hFXIII) immobilizedthereon, the solid support being differentiable from the solid supportshaving binding members immobilized thereon; (c) contacting the solidsupports with a binding agent having binding affinity for hFXIII andbinding agents having binding affinity for each of the analytes, underconditions promoting binding of the binding agents to the analytes onthe solid supports; (d) quantifying the binding agents bound to thesolid supports, and correlating the quantity of binding agents with thedifferentiation parameters to obtain values individually representativeof levels of the analytes and of hFXIII; and (e) normalizing the valuesof the levels representative of the analytes to the value representativeof hFXIII, thereby correcting for variations and decreasing thecoefficient of variation in sample processing or biological matrixeffects during performance of the method, wherein the normalizingcomprises dividing the values of the levels representative of theanalytes by the value representative of hFXIII.
 2. The method of claim1, wherein hFXIII does not bind an analyte from the biological sample.3. The method of claim 1, wherein the binding agent is conjugated to alabel.
 4. The method of claim 3, wherein the label is selected from thegroup consisting of biotin, a fluorescent moiety, phycoerythrin, andhorse radish peroxidase (HRP).
 5. The method of claim 1, wherein thequantifying step further comprises contacting the solid support with alabeled binding moiety, and detecting the amount of label bound to thesolid support.
 6. The method of claim 5, wherein the labeled bindingmoiety comprises streptavidin or avidin.
 7. The method of claim 1,wherein the solid support is selected from a particle, a bead, amagnetic bead, a membrane, a glass surface, and a plastic surface. 8.The method of claim 1, further comprising incubating the sample with asolid support having an antibody to hFXIII immobilized thereon.
 9. Themethod of claim 1, further comprising incubating the sample with a solidsupport having tetramethylrhodamine cadaverine (TMRC) immobilizedthereon.
 10. The method of claim 1, wherein the biological samplecomprises plasma or serum.
 11. A method for normalizing assay results,the method comprising: (a) incubating a biological sample with one ormore solid supports having binding members immobilized thereon thatspecifically bind an analyte in the biological sample, the solidsupports being divided into subpopulations that are differentiable fromeach other by a differentiation parameter comprising a characteristicthat is independent of the binding members immobilized on the solidsupports, the binding members immobilized on each subpopulation bindingto only one analyte in the sample, and performing the incubation underconditions promoting binding of the analytes if present in the sample tothe solid-support-immobilized binding members; (b) incubating thebiological sample with a solid support having hFXIII immobilizedthereon, the solid support being differentiable from the solid supportshaving binding members immobilized thereon; (c) recovering the solidsupports from the sample and incubating the solid supports so recoveredwith biotinylated conjugates having binding affinity for hFXIII andbiotinylated conjugates having binding affinity for each of theanalytes, under conditions promoting binding of the biotinylatedconjugates to hFXIII and to the analytes, respectively, on the solidsupports; (d) recovering the solid supports from step (c) and incubatingthe solid supports so recovered with a labeled binding member selectedfrom streptavidin and avidin; and (e) recovering the solid supports fromstep (d) and detecting label bound to the solid supports thus recovered,correlating the label so detected with the differentiation parameters toobtain values individually representative of levels of the analytes andof hFXIII, and normalizing the values of the levels representative ofthe analytes to the value representative of hFXIII, thereby correctingfor variations and decreasing the coefficient of variation in sampleprocessing or biological matrix effects during performance of themethod, wherein the normalizing comprises dividing the values of thelevels representative of the analytes by the value representative ofhFXIII.
 12. The method of claim 11, wherein the analyte is selected fromhuman immunodeficiency virus (HIV) viral protein p24 antigen, antibodiesto HIV-1, antibodies to HIV-2, antibodies to HIV-1 group O, andantibodies to HIV-1 group M.
 13. The method of claim 1, wherein thebinding members are selected from peptides that bind antibodies toHIV-1, HIV-2, and HIV-O, and antibodies that bind HIV peptides.
 14. Themethod of claim 1, wherein the binding members are selected fromanti-p24 antibodies, gp160, RVTAIEKYLQDQARLNSWGCAFRQVC (SEQ ID NO:5),LNQQRLLNSWGCKGRLVCYTSV (SEQ ID NO:2), and antibodies to human FactorXIII.
 15. The method of claim 11, wherein the biotinylated conjugatesare selected from peptides comprising the amino acid sequencesRILAVERYLKDQQLLGIWGCSGKLICTTAVPFN (SEQ ID NO:1),KYLQDQARLNSWGCAFRQVCHTTVPFVN (SEQ ID NO:4), LNQQRLLNSWGCKGRLVCYTSV (SEQID NO:2), or RVTAIEKYLQDQARLNSWGCAFRQVC (SEQ ID NO:5), or antibodies tohuman Factor XIII.
 16. The method of claim 1, wherein the characteristicis selected from particle size, particle diameter, particle composition,light scatter, absorbance, number of particles, fluorescent dyes orcolored dyes that impart different emission spectra and/or scatteringcharacteristics to the solid supports, or from different concentrationsof one or more fluorescent dyes.
 17. The method of claim 16, wherein thecharacteristic is determined by flow cytometry.